Hepatocellular carcinoma-related genes and polypeptides, and method for detecting hepatocellular carcinomas

ABSTRACT

Genes up-regulated in hepatocellular carcinomas and polypeptides encoded by these genes are provided. Vectors, transformants and methods for producing the recombinant polypeptides are also provided. Probes and primers of these genes and antibodies against the polypeptides are also provided. The probes, primers and antibodies can be used as reagents for detecting hepatocellular carcinomas. Methods for detecting hepatocellular carcinomas using such detection reagents are further provided. Antisense nucleotide sequences of these genes are also provided and can be used to inhibit growth of hepatocellular carcinomas.

RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.10/490,605, filed Sep. 15, 2004 now U.S. Pat. No. 7,345,156 as anational stage application, filed under 35 U.S.C. §371, of InternationalApplication No. PCT/JP02/09873, filed on Sep. 25, 2002, which claims thebenefit of Canadian Patent Application 2,399,569, filed Aug. 23, 2002and U.S. Ser. No. 60/324,261, filed Sep. 25, 2001.

TECHNICAL FIELD

The present invention relates to genes up-regulated in hepatocellularcarcinomas, polypeptides encoded by the genes, and a method fordetecting hepatocellular carcinomas.

BACKGROUND ART

cDNA microarray technologies have enabled one to obtain comprehensiveprofiles of gene expression in normal versus malignant cells (Perou, C.M. et al., Nature. 406: 747-752, 2000; Clark, E. A. et al., Nature. 406:532-535, 2000; Okabe, H. et al., Cancer Res. 61: 2129-2137, 2001). Thisapproach discloses the complex nature of cancer cells, and helps toimprove understanding of carcinogenesis. Identification of genes thatare deregulated in tumors can lead to more precise and accuratediagnosis of individual cancers, and to development of novel therapeutictargets (Golub, T. R. et al., Science 286: 531-537, 1999).

Hepatocellular carcinoma (HCC) is a leading cause of cancer deathsworldwide. In spite of recent progress in therapeutic strategies,prognosis of patients with advanced HCC remains very poor. Althoughmolecular studies have revealed that alterations of TP53, CTNNB1 and/orAXIN1 genes can be involved in hepatocarcinogenesis (Perou, C. M. etal., Nature. 406: 747-752, 2000; Satoh, S. et al., Nat. Genet. 24:245-250, 2000), these changes appear to be implicated in only a fractionof HCCs. Accordingly, a ultimate gene that can be a novel diagnosticmarker and/or drug target for treatment of cancers has been desired.

The present inventors previously reported that a novel gene, VANGL1, wasidentified by genome-wide analysis of HCCs (Yagyu, R. et al.,International Journal of Oncology 20: 1173-1178, 2002).

DISCLOSURE OF THE INVENTION

An objective of the present invention is to provide genes up-regulatedin hepatocellular carcinomas, polypeptides encoded by the genes, and amethod for detecting hepatocellular carcinomas.

The present inventors have analyzed expression profiles of HCCs by meansof a cDNA microarray representing 23,040 genes. These efforts havepinpointed 165 genes, including 69 ESTs, which appear to be up-regulatedfrequently in cancer tissues compared with corresponding non-cancerousliver cells. The inventors isolated three genes from among thetranscripts whose expression was frequently elevated in HCCs. Thesegenes encode products that shared structural features withcentaurin-family proteins.

One of the three genes corresponds to an EST, Hs.44579 of a UniGenecluster, and was found to be a novel gene over-expressed at chromosomalband 1p36.13. Since an open reading frame of this gene encoded a proteinapproximately 60% identical to that of development and differentiationenhancing factor 2 (DDEF2), the inventors termed this gene developmentand differentiation enhancing factor-like 1 (DDEFL1).

Another gene up-regulated in HCCs corresponds to an EST (Hs. 122730) ofa UniGene cluster. The predicted amino acid sequence shared 40% and 63%identity with strabismus (Van Gogh), which is involved in cell polarityand cell fate decisions in Drosophila, and Van Gogh Like 2 (VANGL2).Hence, this gene was termed Van Gogh Like 1 (VANGL1).

Another gene up-regulated in HCCs was found to be LGN (GenBank accessionnumber U54999). LGN protein interacts with alpha subunit of inhibitoryheterotrimeric G proteins (Gα₁₂).

Gene transfer of DDEFL1 or LGN promoted proliferation of cells thatlacked endogenous expression of either of these genes. Furthermore,reduction of DDEFL1, VANGL1 or LGN expression by transfection of theirspecific anti-sense S-oligonucleotides inhibited the growth ofhepatocellular carcinoma cells.

The above findings would contribute to clarify the mechanisms of HCC andto develop new strategies for diagnosis and treatment of HCC.

The present invention specifically provides

(1) an isolated nucleic acid selected from the group consisting of:

(a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 orNO: 3;

(b) a nucleic acid encoding a polypeptide comprising the amino acidsequence of SEQ ID NO: 2 or NO: 4;

(c) a nucleic acid comprising a strand that hybridizes under highstringent conditions to a nucleotide sequence consisting of SEQ ID NO: 1or NO: 3 or the complement thereof,

(2) an isolated polypeptide selected from the group consisting of:

(a) a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 orNO: 3;

(b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 orNO: 4;

(c) a polypeptide having at least 65% identity to SEQ ID NO: 2 or NO: 4,

(3) a vector carrying the nucleic acid of (1),

(4) a transformant carrying the nucleic acid of (1) or the vector of(3),

(5) a method of producing a polypeptide, the method comprising culturingthe transformant of (4) in a culture, expressing the polypeptide in thetransformant, and recovering the polypeptide from the culture,

(6) an antibody that specifically binds to the polypeptide of (2),

(7) a method for detecting hepatoceullar carcinoma, the methodcomprising the steps of:

(a) preparing a biological sample from a subject;

(b) measuring the expression level of at least one of polypeptidesselected from the group consisting of the polypeptide of SEQ ID NO: 1, apolypeptide of SEQ ID NO: 3, and the polypeptide of SEQ ID NO: 5;

(c) comparing the expression level with that measured in a non-canceroussample; and

(d) determining the presence or absence of the cancer in the subject,

(8) a reagent for detecting hepatocellular carcinomas, comprising anucleic acid comprising a strand that hybridizes under high stringentconditions to a nucleotide sequence consisting of SEQ ID NO: 1, NO: 3,or NO: 5 or the complement thereof,

(9) a reagent for detecting hepatocellular carcinomas, comprising theantibody of (6), and

(10) a method for inhibiting growth of hepatocellular carcinomas, themethod comprising introducing at least one of antisense oligonucleotidesthat hybridizes with the nucleotide sequence of SEQ ID NO: 1, NO: 3, orNO: 5 into hepatocelluar carcinomas.

The present invention will be illustrated below in more detail.

Nucleic Acids

The present invention provides genes up-regulated in hepatocellularcarcinomas.

The nucleotide sequence and the amino acid sequence of DDEFL1 are shownas SEQ ID NO: 1 and NO: 2, respectively. The complete cDNA of DDEFL1consisted of 4050 nucleotides, with an open reading frame of 2712nucleotides encoding a 903-amino-acid protein (GenBank accession numberAB051853). The amino acid sequence of DDFEL1 showed 60% identity toDDEFL2 and 46% identity to DDEF/ASAP1, and contained an ArfGTPase-activating protein (ArfGAP) domain and two ankyrin repeats.

DDEFL1 showed 60% identity to a member of the centaurin family, DDEF2, aprotein that regulates re-organization of the actin cytoskeleton. Thissuggests that DDEFL1 may also play a role in organization of cellularstructure (Randazzo, P. A. et al., The Arf GTPase-activating proteinASAP1 regulates the actin cytoskeleton, Proc. Natl. Acad. Sci. USA 97:4011-4016, 2000). Because DDEFL1 also conserves a PH domain and anArfGAP motif it appears to be a new member of the centaurin family,regulating Arf small GTPase by means of GAP activity. The PH domain,observed in the majority of molecules belonging to the Dbl family ofGEFs, is thought to play a crucial role in relocation of proteins byinteracting with specific target molecules and/or by directly regulatingcatalytic domains (Jackson, T. R. et al., Trends Biochem Sci. 25:489-495, 2000; Cerione, R. A. and Zheng, Y., Curr. Opin. Cell. Biol. 8:216-222, 1996; Chardin, P. et al., Nature 384: 481-484, 1996). AlthoughDDEF2 is localized in peripheral focal adhesions, the inventors foundmyc-tagged DDEFL1 protein to be diffuse in cytoplasm.

Arf proteins have been implicated in important cellular processes suchas vesicular membrane transport, maintenance of the integrity of ER andGolgi compartments, and regulation of the peripheral cytoskeleton(Cukierman, E. et al., Science 270: 1999-2002, 1995). Six members of Arffamily (Arf1-Arf6) and their functions have been identified so far(Moss, J. and Vaughan, M., J. Biol. Chem. 270: 12327-12330, 1995). Forexample, Arf6 proteins have been implicated as regulators of thecytoskeleton to alter the morphology of focal adhesions and to blockspreading of cells, and DDEF2 displays GAP activity toward Arf1.

Over-expression of DDEFL1 promoted growth promotion and survival ofcells under low-serum conditions. This suggests that DDEFL1 may providea growth advantage to cancer cells in poor nutritional and hypoxicconditions. The frequent up-regulation of DDEFL1 in HCCs underscores theimportance of this gene in hepatocarcinogenesis.

The nucleotide sequence and the amino acid sequence of VANGL1 are shownas SEQ ID NO: 3 and NO: 4, respectively. The determined cDNA sequenceconsisted of 1879 nucleotides containing an open reading frame of 1572nucleotides encoding a 524-amino-acid protein (GenBank accession numberAB057596).

Strabismus (stbm) was identified as a gene responsible for a mutantfruit fly with rough eye phenotype (Wolff T. and Rubin G. M.,Development 125:1149-1159, 1998). The gene is required to maintainpolarity in the eye, legs and bristles and to decide cell fate of R3 andR4 photoreceptors in the Drosophila. A mouse gene homologous to stbm,Ltap, was altered in the neural tube mutant mouse Loop-tail, which is ahuman model of neural tube defects (NTDs) (Kibar Z et al., Nat. Genet.28: 251-255, 2001). Hence, VANGL1 may also play important roles incellular polarity, cell fate decision, and/or organization of tissues.Since VANGL1 is frequently up-regulated in HCCs and suppression of itsexpression significantly reduced growth or survival of cancer cells,VANGL1 may confer prolonged survival and/or depolarized growth to cancercells.

The nucleotide sequence and the amino acid sequence of LGN are shown asSEQ ID NO: 5 and NO: 6, respectively. LGN cDNA consists of 2336nucleotides and encodes a 677 amino acid peptide.

LGN protein was previously reported as a protein interacting with alphasubunit of inhibitory heterotrimeric G proteins (Gαi2) (Mochizuki, N. etal., Gene 181: 39-43, 1996). The activating mutations of Gαi2 have everbeen reported in pituitary tumor and other endocrine tumors (Hermouet,S. et al., Proc. Natl. Acad. Sci. USA 88: 10455-10459, 1991; Pace, A. M.et al., Proc. Natl. Acad. Sci. USA. 88: 7031-7035, 1991; Lyons, J. etal, Science 249: 655-659, 1990). However, involvement of LGN intumorigenesis or carcinogenesis has not yet been reported. Colonyformation assay suggested that LGN might have oncogenic activity.Enhanced expression of LGN may activate Gαi2 and mediate oncogenicsignals in hepatocarcinogenesis.

The nucleic acid of the present invention includes cDNA, genomic DNA,chemically synthesized DNA, and RNA. It may be single-stranded ordouble-stranded.

The “isolated nucleic acid” used herein means a nucleic acid thestructure of which is not identical to that of any naturally occurringnucleic acid or to that of any fragment of a naturally occurring genomicnucleic acid spanning more than three separate genes. The term thereforeincludes, for example, (a) a DNA which has the sequence of part of anaturally occurring genomic DNA molecule in the genome of the organismin which it naturally occurs; (b) a nucleic acid incorporated into avector or into the genomic DNA of a prokaryote or eukaryote in a mannersuch that the resulting molecule is not identical to any naturallyoccurring vector or genomic DNA; (c) a separate molecule such as a cDNA,a genomic fragment, a fragment produced by polymerase chain reaction(PCR), or a restriction fragment; and (d) a recombinant nucleotidesequence that is part of a hybrid gene, i.e., a gene encoding a fusionprotein. Specifically excluded from this definition are nucleic acids ofDNA molecules present in mixtures of different (i) DNA molecules, (ii)transfected cells, or (iii) cell clones; e.g., as these occur in a DNAlibrary such as a cDNA or genomic DNA library.

In one embodiment, the nucleic acid of the present invention includes anucleic acid comprising the nucleotide sequence of DDEFL1 or VANGL1,specifically SEQ ID NO: 1 or NO: 3.

In another embodiment, the nucleic acid of the present inventionincludes a nucleic acid encoding a polypeptide comprising the amino acidsequence of DDEFL1 or VANGL1, specifically, SEQ ID NO: 2 or NO: 4. Thus,the nucleic acid comprising arbitrary sequences based on the degeneracyof the genetic code are included.

In still another embodiment, the nucleic acid of the present inventionincludes a variant nucleic acid of SEQ ID NO: 1 or NO: 3. The variantincludes a nucleic acid comprising a strand that hybridizes under highstringent conditions to a nucleotide sequence consisting of SEQ ID NO: 1or NO: 3 or the complement thereof.

The term “complement” used herein means one strand of a double-strandednucleic acid, in which all the bases are able to form base pairs with asequence of bases in another strand. Also, “complementary” is defined asnot only those completely matching within a continuous region of atleast 15 contiguous nucleotides, but also those having identity of atleast 65%, preferably 70%, more preferably 80%, still more preferably90%, and most preferably 95% or higher within that region.

As used herein, “percent identity” of two nucleic acids is determinedusing the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA87: 2264-2268, 1990) modified as in Karlin and Altschul (Proc. Natl.Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol.215:403-410, 1990). BLAST nucleotide searches are performed with theNBLAST program, score=100, wordlength=12. Homology search of protein canreadily be performed, for example, in DNA Databank of JAPAN (DDBJ), byusing the FASTA program, BLAST program, etc. BLAST protein searches areperformed with the XBLAST program, score=50, wordlength=3. Where gapsexist between two sequences, Gapped BLAST is utilized as described inAltsuchl et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When utilizingBLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) are used.

Preferably, the variant includes a nucleotide sequence that is at least65% identical to the nucleotide sequence shown in SEQ ID NO: 1 or NO: 3.More preferably, the variant is at least 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or more, identical to the nucleotide sequenceshown in SEQ ID NO: 1 or NO: 3. In the case of a variant which is longerthan or equivalent in length to the reference sequence, e.g., SEQ ID NO:1 or NO: 3, the comparison is made with the full length of the referencesequence. Where the variant is shorter than the reference sequence,e.g., shorter than SEQ ID NO: 1 or NO: 3, the comparison is made tosegment of the reference sequence of the same length (excluding any looprequired by the homology calculation)

The stringency of hybridization is defined as equilibrium hybridizationunder the following conditions: 42° C., 2×SSC, 0.1% SDS (lowstringency); 50° C., 2×SSC, 0.1% SDS (medium stringency); and 65° C.,2×SSC, 0.1% SDS (high stringency) If washings are necessary to achieveequilibrium, the washings are performed with the hybridization solutionfor the particular stringency desired. In general, the higher thetemperature, the higher is the homology between two strands hybridizingat equilibrium.

There is no restriction on length of the nucleic acid of the presentinvention, but it preferably comprises at least 15, 20, 30, 40, 50, 100,150, 200, 300, 400, 500, 1000, 1500, 2000, 2500, or 3000 nucleotides.

The nucleic acid of the present invention includes polynucleotides usedas probes or primers specifically hybridizing with the nucleotidesequence of SEQ ID NO: 1 or NO: 3 or its complement. The term“specifically hybridizing” means that hybridizing under a normalhybridization condition, preferably a stringent condition with thenucleotide sequence of SEQ ID NO: 1 or NO: 3, but not crosshybridizingwith DNAs encoding other polypeptides.

The primers and probes comprise at least 15 continuous nucleotideswithin the nucleotide sequence of SEQ ID NO: 1 or 3 or complementary tothe sequence. In general, the primers comprises 15 to 100 nucleotides,and preferably 15 to 35 nucleotides, and the probes comprise at least 15nucleotides, preferably at least 30 nucleotides, containing at least aportion or the whole sequence of SEQ ID NO: 1 or NO: 3. The primers canbe used for amplification of the nucleic acid encoding the polypeptideof the present invention and the probes can be used for the isolation ordetection of the nucleic acid encoding the polypeptide of the presentinvention. The primers and probes of the present invention can beprepared, for example, by a commercially available oligonucleotidesynthesizing machine. The probes can be also prepared as double-strandedDNA fragments which are obtained by restriction enzyme treatments andthe like.

The nucleic acid of the present invention includes an antisenseoligonucleotide that hybridizes with any site within the nucleotidesequence of SEQ ID NO: 1 or 3. The term “antisense oligonucleotides” asused herein means, not only those in which the entire nucleotidescorresponding to those constituting a specified region of a DNA or mRNAare complementary, but also those having a mismatch of one or morenucleotides, as long as DNA or mRNA and an oligonucleotide canspecifically hybridize with the nucleotide sequence of SEQ ID NO: 1 orNO: 3.

The antisense oligonucleotide is preferably that against at least 15continuous nucleotides in the nucleotide sequence of SEQ ID NO: 1 or NO:3. The above-mentioned antisense oligonucleotide, which contains aninitiation codon in the above-mentioned at least 15 continuousnucleotides, is even more preferred.

The antisense oligonucleotides of the present invention includes analogscontaining lower alkyl phosphonate (e.g., methyl-phosphonate orethyl-phosphonate), phosphothioate, and phosphoamidate.

The antisense oligonucleotide of the present invention, acts upon cellsproducing the polypeptide of the invention by binding to the DNA or mRNAencoding the polypeptide and inhibits its transcription or translation,promotes the degradation of the mRNA, inhibiting the expression of thepolypeptide of the invention.

The nucleic acid of the present invention can be prepared as follows.cDNA encoding the polypeptide of the present invention can be prepared,for example, by preparing a primer based on nucleotide information (forexample, SEQ ID NO: 1 or NO: 3) of DNA encoding the polypeptide of thepresent invention and performing plaque PCR (Affara NA et al. (1994)Genomics 22, 205-210). Genomic DNA can be prepared, for example, by themethod using commercially available “Qiagen genomic DNA kits” (Qiagen,Hilden, Germany). The nucleotide sequence of the DNA acquired can bedecided by ordinary methods in the art by using, for example, thecommercially available “dye terminator sequencing kit” (AppliedBiosystems). The nucleic acid of the present invention, as stated later,can be utilized for the production of a recombinant protein anddetection of hepatocellular carcinoma.

Vectors, Transformants, and Production of Recombinant Polypeptide

The present invention also features a vector into which the nucleic acidof the present invention has been inserted.

The vector of the present invention includes a vector for preparing therecombinant polypeptide of the present invention. Any vector can be usedas long as it enables expression of the polypeptide of the presentinvention.

Examples of the expression vector include bacterial (e.g. Escherichiacoli) expression vectors, yeast expression vectors, insect expressionvectors, and mammalian expression vectors.

In the present invention, mammalian expression vectors such aspcDNA3.1-myc/H is or pcDNA 3.1 vector (Invitrogen) can be used.Insertion of the nucleic acid of the present invention into a vector canbe done using ordinary methods in the art.

The vector of the present invention also includes a vector forexpressing the polypeptide of the present invention in vivo (especiallyfor gene therapy). Various viral vectors and non-viral vectors can beused as long as they enable expression of the polypeptide of the presentinvention in vivo. Examples of viral vectors are adenovirus vectors,retrovirus vectors, etc. Cationic liposomes can be given as examples ofnon-viral vectors.

The present invention also provides a transformant carrying, in anexpressible manner, the nucleic acid of the present invention. Thetransformant of the present invention includes, those carrying theabove-mentioned expression vector into which nucleic acid of the presentinvention has been inserted, and those having host genomes into whichthe nucleic acid of the present invention has been integrated. Thenucleic acid of the invention is retained in the transformant in anyform as long as the transformant can express the nucleic acid.

There is no particular restriction as to the cells into which the vectoris inserted as long as the vector can function in the cells to expressthe nucleic acid of the present invention. For example, E. coli, yeast,mammalian cells and insect cells can be used as hosts. Preferably,mammalian cells such as COS7 cells and NIH3T3 cells. Introduction of avector into a cell can be done using known methods such aselectroporation and calcium phosphate method.

Common methods applied in the art may be used to isolate and purify saidrecombinant polypeptide from the transformant. For example, aftercollecting the transformant and obtaining the extracts, the objectivepolypeptide can be purified and prepared by, ion exchangechromatography, reverse phase chromatography, gel filtration, oraffinity chromatography where an antibody against the polypeptide of thepresent invention has been immobilized in the column, or by combiningseveral of these columns.

Also when the polypeptide of the present invention is expressed withinhost cells (for example, animal cells, E. coli) as a fusion protein withglutathione-S-transferase protein or as a recombinant polypeptidesupplemented with multiple histidines, the expressed recombinantpolypeptide can be purified using a glutathione column or nickel column.After purifying the fusion protein, it is also possible to excluderegions other than the objective polypeptide by cutting with thrombin orfactor-Xa as required.

Polypeptides

The present invention provides isolated polypeptides encoded by DDEFL1or VANGL1 (e.g. SEQ ID NO: 1 or NO: 3). In specific embodiments, thepolypeptides of the present invention includes a polypeptide encoded bythe nucleotide sequence of SEQ ID NO: 1 or NO: 3 and a polypeptidecomprising the amino acid sequence of SEQ ID NO: 2 or NO: 4.

The “isolated polypeptide” used herein means a polypeptide that issubstantially pure and free from other biological macromolecules. Thesubstantially pure polypeptide is at least 75% (e.g., at least 80, 85,95, or 99%) pure by dry weight. Purity can be measured by anyappropriate standard method, for example by column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis.

The polypeptide of the present invention includes variants of SEQ ID NO:2 or NO: 4 as long as the variants are at least 65% identical to SEQ IDNO: 2 or NO: 4. The variants may be a polypeptide comprising the aminoacid sequence of SEQ ID NO: 2 or NO: 4 in which one or more amino acidshave been substituted, deleted, added, and/or inserted. The variants mayalso be a polypeptide encoded by a nucleic acid comprising a strand thathybridizes under high stringent conditions to a nucleotide sequenceconsisting of SEQ ID NO: 1 or NO: 3.

Polypeptides having amino acid sequences modified by deleting, addingand/or replacing one or more amino acid residues of a certain amino acidsequence, have been known to retain the original biological activity(Mark, D. F. et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666,Zoller, M. J. &Smith, M., Nucleic Acids Research (1982) 10, 6487-6500,Wang, A. et al., Science 224, 1431-1433, Dalbadie-McFarland, G. et al.,Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413).

The number of amino acids that are mutated by substitution, deletion,addition, and/or insertion is not particularly restricted. Normally, itis 10% or less, preferably 5% or less, and more preferably 1% or less ofthe total amino acid residues.

As for the amino acid residue to be mutated, it is preferable to bemutated into a different amino acid in which the properties of the aminoacid side-chain are conserved. Examples of properties of amino acid sidechains are, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V),hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and aminoacids comprising the following side chains: an aliphatic side-chain (G,A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); asulfur atom containing side-chain (C, M); a carboxylic acid and amidecontaining side-chain (D, N, E, Q); a base containing side-chain (R, K,H); and an aromatic containing side-chain (H, F, Y, W) (The parentheticletters indicate the one-letter codes of amino acids). A “conservativeamino acid substitution” is a replacement of one amino acid belonging toone of the above groups with another amino acid in the same group.

A deletion variant includes a fragment of the amino acid sequence of SEQID NO: 1 or NO: 3. The fragment is a polypeptide having an amino acidsequence which is partly, but not entirely, identical to the abovepolypeptides of this invention. The polypeptide fragments of thisinvention usually consist of 8 amino acid residues or more, andpreferably 12 amino acid residues or more (for example, 15 amino acidresidues or more). Examples of preferred fragments include truncationpolypeptides, having amino acid sequences lacking a series of amino acidresidues including either the amino terminus or carboxyl terminus, ortwo series of amino acid residues, one including the amino terminus andthe other including the carboxyl terminus. Furthermore, fragmentsfeatured by structural or functional characteristics are alsopreferable, which include those having. α-helix and α-helix formingregions, β-sheet and β-sheet forming regions, turn and turn formingregions, coil and coil forming regions, hydrophilic regions, hydrophobicregions α-amphipathic regions, β-amphipathic regions, variable regions,surface forming regions, substrate-binding regions, and highantigenicity index region. Biologically active fragments are alsopreferred. Biologically active fragments mediate the activities of thepolypeptides of this invention, which fragments include those havingsimilar or improved activities, or reduced undesirable activities. Forexample, fragments having the activity to transduce signals into cellsvia binding of a ligand, and furthermore, fragments having antigenicityor immunogenicity in animals, especially humans are included. Thesepolypeptide fragments preferably retain the antigenicity of thepolypeptides of this invention.

Further, an addition variant includes a fusion protein of thepolypeptide of the present invention and another peptide or polypeptide.Fusion proteins can be made by techniques well known to a person skilledin the art, such as by linking the DNA encoding the polypeptide of theinvention with DNA encoding other peptides or polypeptides, so as theframes match, inserting this into an expression vector and expressing itin a host. There is no restriction as to the peptides or polypeptidesfused to the polypeptide of the present invention.

Known peptides, for example, FLAG (Hopp, T. P. et al., Biotechnology(1988) 6, 1204-1210), 6×His containing six H is (histidine) residues,10× His, Influenza agglutinin (HA), human c-myc fragment, VSP-GPfragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigenfragment, lck tag, α-tubulin fragment, B-tag, Protein C fragment, andsuch, can be used as peptides that are fused to the polypeptide of thepresent invention. Examples of polypeptides that are fused topolypeptide of the invention are, GST (glutathione-S-transferase),Influenza agglutinin (HA), immunoglobulin constant region,β-galactosidase, MBP (maltose-binding protein), and such.

Fusion proteins can be prepared by fusing commercially available DNAencoding these peptides or polypeptides with the DNA encoding thepolypeptide of the present invention and expressing the fused DNAprepared.

The variant polypeptide is preferably at least 65% identical to theamino acid sequence shown in SEQ ID NO: 2 or NO: 4. More specifically,the modified polypeptide is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or more, identical to the amino acid sequence shownin SEQ ID NO: 2 or NO: 4. In the case of a modified polypeptide which islonger than or equivalent in length to the reference sequence, e.g. SEQID NO: 2 or NO: 4, the comparison is made with the full length of thereference sequence. Where the modified polypeptide is shorter than thereference sequence, e.g., shorter than SEQ ID NO: 2 or NO: 4, thecomparison is made to segment of the reference sequence of the samelength.

As used herein, “percent identity” of two amino acid sequences isdetermined in the same manner as described above for the nucleic acids.

The polypeptide of the present invention can be prepared by methodsknown to one skilled in the art, as a natural polypeptide or arecombinant polypeptide made using genetic engineering techniques asdescribed above. For example, a natural polypeptide can be obtained bypreparing a column coupled with an antibody obtained by immunizing asmall animal with the recombinant polypeptide, and performing affinitychromatography for extracts of liver tissues or cells expressing highlevels of the polypeptide of the present invention. A recombinantpolypeptide can be prepared by inserting DNA encoding the polypeptide ofthe present invention (for example, DNA comprising the nucleotidesequence of SEQ ID NO: 1 or 3) into a suitable expression vector,introducing the vector into a host cell, allowing the resultingtransformant to express the polypeptide, and recovering the expressedpolypeptide.

The variant polypeptide can be prepared, for example, by inserting amutation into the amino acid sequence of SEQ ID NO: 1 or NO: 3 by aknown method such as the PCR-mediated, site-directed-mutation-inductionsystem (GIBCO-BRL, Gaithersburg, Md.), oligonucleotide-mediated,sight-directed-mutagenesis (Kramer, W. and Fritz, H J (1987) Methods inEnzymol. 154:350-367).

Antibodies

The present invention also features an antibody that specifically bindsto the polypeptide of the present invention. There is no particularrestriction as to the form of the antibody of the present invention andinclude polyclonal antibodies and monoclonal antibodies. The antiserumobtained by immunizing animals such as rabbits with the polypeptide ofthe present invention, polyclonal and monoclonal antibodies of allclasses, humanized antibodies made by genetic engineering, humanantibodies, are also included.

Polyclonal antibodies can be made by, obtaining the serum of smallanimals such as rabbits immunized with the polypeptide of the presentinvention, attaining a fraction recognizing only the polypeptide of theinvention by an affinity column coupled with the polypeptide of thepresent invention, and purifying immunoglobulin G or M from thisfraction by a protein G or protein A column.

Monoclonal antibodies can be made by immunizing small animals such asmice with the polypeptide of the present invention, excising the spleenfrom the animal, homogenizing the organ into cells, fusing the cellswith mouse myeloma cells using a reagent such as polyethylene glycol,selecting clones that produce antibodies against the polypeptide of theinvention from the fused cells (hybridomas), transplanting the obtainedhybridomas into the abdominal cavity of a mouse, and extracting ascites.The obtained monoclonal antibodies can be purified by, for example,ammonium sulfate precipitation, protein A or protein G column, DEAE ionexchange chromatography, or an affinity column to which the polypeptideof the present invention is coupled. The antibody of the invention canbe used for purifying and detecting the polypeptide of the invention. Inparticular, it can be used for detecting hepatocellular carcinoma.

The human antibodies or humanized antibodies can be prepared by methodscommonly known to one skilled in the art. For example, human antibodiescan be made by, immunizing a mouse whose immune system has been changedto that of humans, with the polypeptide of the present invention. Also,humanized antibodies can be prepared by, for example, cloning theantibody gene from monoclonal antibody producing cells and using the wCDR graft method which transplants the antigen-recognition site of thegene into a known human antibody.

Detection Methods

The present invention further provides a method of detectinghepatocellular carcinoma using the DDEFL1, VANGL1, or LGN polypeptide asa marker.

The detection can be performed by measuring an expression level of atleast one of DDEFL1, VANGL1, and LGN polypeptides in a biological samplefrom a subject, comparing the expression level with that in anon-cancerous sample, and determining the presence or absence of thecancer in a subject.

A biological sample used herein include any liver tissues or cellsobtained from a subject who is in need of detection of hepatocellularcarcinoma. In particular, liver biopsy specimen can be used. Thebiological sample also includes an mRNA, cRNA or cDNA sample preparedfrom liver tissues or cells. mRNA and cDNA samples can be prepared by aconventional method. cRNA refers to RNA transcribed from a template cDNAwith RNA polymerase. cRNA can be synthesized from T7 promoter-attachedcDNA as a template by using T7 RNA polymerase. A commercially availablecRNA transcription kit for DNA chip-based expression profiling can beused.

In specific embodiments, the expression level of DDEFL1, VANGL1 or LGNpolypeptide can be measured in the RNA, cDNA, or polypeptide level.

The mRNA expression level can be measured by, for example, a Northernblotting method using a probe that hybridizes with the nucleotidesequence of DDEFL1, VANGL1, or LGN, an RT-PCR method using a primer thathybridizes with the nucleotide sequence of DDEFL1, VANGL1, or LGN, andsuch.

The probes or primers used in the detection method of the presentinvention include a nucleic acid specifically hybridizing with thenucleotide sequence of SEQ ID NO: 1, NO: 3, or NO: 5, or its complement.The term “specifically hybridizing” means that hybridizing under anormal hybridization condition, preferably a stringent condition withthe nucleotide sequence of SEQ ID NO: 1, NO: 3, or NO: 5, but notcrosshybridizing with DNAs encoding other polypeptides.

The primers and probes comprise at least 15 continuous nucleotideswithin the nucleotide sequence of SEQ ID NO: 1, NO: 3, or NO: 5 orcomplementary to the sequence. In general, the primers comprises 15 to100 nucleotides, and preferably 15 to 35 nucleotides, and the probescomprise at least 15 nucleotides, preferably at least 30 nucleotides,containing at least a portion or the whole sequence of SEQ ID NO: 1, NO:3, or NO: 5. The primers and probes can be prepared, for example, by acommercially available oligonucleotide synthesizing machine. The probescan be also prepared as double-stranded DNA fragments which are obtainedby restriction enzyme treatments and the like.

The cDNA expression level can be measured by, for example, a methodutilizing a DNA array (Masami Muramatsu and Masashi Yamamoto, NewGenetic Engineering Handbook pp. 280-284, YODOSHA Co., LTD.).Specifically, first, a cDNA sample prepared from a subject and a solidsupport, on which polynucleotide probes hybridizing with the nucleotidesequence of DDEFL1, VANGL1, or LGN are fixed, are provided. As theprobes, those as described above can be used. Plural kinds of probes canbe fixed on the solid support in order to detect plural kinds of targetpolynucleotides. The cDNA sample is labeled for detection according toneeds. The label is not specifically limited so long as it can bedetected, and includes, for example, fluorescent labels, radioactivelabels, and so on. The labeling can be carried out by conventionalmethods (L. Luo et al., “Gene expression profiles of laser-capturedadjacent neuronal subtypes”, Nat. Med. (1999) pp. 117-122).

The cDNA sample is then contacted with the probes on the solid supportto allow the cDNA sample to hybridize with the probes. Although thereaction solution and the reaction condition for hybridization variesdepending on various factors, such as the length of the probe, they canbe determined according to usual methods well known to those skilled inthe art.

The intensity of hybridization between the cDNA sample and the probes onthe solid support is measured depending on the kind of the label of thecDNA sample. For example, a fluorescent label can be detected by readingout the fluorescent signal with a scanner.

The hybridization intensity of the test cDNA sample and the control cDNAsample (e.g. cDNA from non-cancerous tissues or cells) can be measuredsimultaneously in one measurement by labeling them with differentfluorescent labels. For example, one of the above-mentioned cDNA samplescan be labeled with Cy5, and the other with Cy3. The intensity of Cy5and Cy3 fluorescent signals show the expression level of the respectivecDNA samples (Duggan et al., Nat. Genet. 21:10-14, 1999).

In this method, cRNA can be measured in place of cDNA.

Furthermore, the polypeptide expression level can be measured using anantibody against DDEFL1, VANGL1, or LGN polypeptide by, for example, SDSpolyacrylamide electrophoresis, Western blotting, dot-blotting,immunoassay such as immunoprecipitation, fluoroimmunoassay,radioimmunoassay, enzyme immunoassay (e.g. enzyme-linked immunosorbentassay (ELISA)), and immunohistochemical staining, etc.

In specific embodiments, a biological sample is contacted with anantibody against DDEFL1, VANGL1, or LGN polypeptide immobilized on asolid support, the antibody-antigen complex on the solid support iscontacted with a second antibody labeled with a detectable label, andthe label is detected by an appropriate method.

The antibody used in the detection method of the present inventionincludes any antibody that binds to the DDEFL1, VANGL1, or LGNpolypeptide, specifically the polypeptide with the amino acid sequenceof SEQ ID NO: 2, NO: 4, or NO: 6, including antiserum obtained byimmunizing animals such as rabbits with the DDEFL1, VANGL1, or LGNpolypeptide, polyclonal and monoclonal antibodies of all classes,humanized antibodies made by genetic engineering, and human antibodies.These antibodies can be prepared as described above.

The expression level measured as described above is compared with thatmeasured in a non-cancerous sample to determine the presence or absenceof hematocellular carcinoma in the subject. When the expression levelmeasured in the sample from the subject is higher than that measured inthe non-cancerous sample, the subject is judged to have the cancer orthe risk of the cancer. On the other hand, the expression level in thesubject sample is not higher compared with that in the non-canceroussample, then, the subject is judged to be free from the cancer.Specifically, whether the expression level in the subject sample ishigher than that in the non-cancerous sample, can be determined based onthe relative expression ratio (subject sample/non-cancerous sample); theexpression level is judged as being higher when the relative expressionratio is more than 2.0.

Detection Reagents

The present invention provides detection reagents for hepatocellularcarcinomas.

In one embodiment, the detection reagent of the present inventioncomprises a polynucleotide having at least 15 nucleotides whichhybridizes with DDEFL1, VANGL1 or LGN, specifically SEQ ID NO: 1, NO: 3,or NO: 5. The polynucleotide can be used in the above-mentioneddetection method of the present invention as a probe or a primer. Whenused as a probe, the polynucleotides contained in the detection reagentof the present invention can be labeled. The method of labelingincludes, for example, a labeling method using T4 polynucleotide kinaseto phosphorylate the 5′-terminus of the polynucleotide with ³²P; and amethod of introducing substrate bases, which are labeled with isotopessuch as ³²P, fluorescent dyes, biotin, and so on using random hexameroligonucleotides and such as primers and DNA polymerase such as Klenowenzyme (the random prime method, etc.).

In another embodiment, the detection reagent of the present inventioncomprises an antibody that binds to the DDEFL1, VANGL1, or LGNpolypeptide, specifically the polypeptide having the amino acid sequenceof SEQ ID NO: 2, NO: 4, or NO: 6. The antibodies are used to detect thepolypeptides of the present invention in the above-mentioned detectionmethod of the present invention. The antibodies may be labeled accordingto the diction method. Furthermore, the antibodies may be immobilized ona solid support.

The detection reagent of the present invention may further comprise amedium or additive, including sterilized water, physiological saline,vegetable oils, surfactants, lipids, solubilizers, buffers, proteinstabilizers (such as bovine serum albumin and gelatin), preservatives,and such, as long as it does not affect the reactions used in thedetection method of the present invention.

Methods for Inhibiting Growth of Hematocellular Carcinomas

The present invention further provides a method for inhibiting growth ofhepatocellular carcinomas. In specific embodiments, this method can beperformed by introducing an antisense oligonucleotide of DDEFL1, VANGL1,or LGN into the target cells.

The antisense oligonucleotide used in this method hybridizes with anysite within the nucleotide sequence of SEQ ID NO: 1, NO: 3, or NO: 5.The antisense oligonucleotides include not only those in which theentire nucleotides corresponding to those constituting a specifiedregion of a DNA or mRNA are complementary, but also those having amismatch of one or more nucleotides, as long as DNA or mRNA and anoligonucleotide can specifically hybridize with the nucleotide sequenceof SEQ ID NO: 1, NO: 3, or NO: 5.

The antisense oligonucleotide is preferably that against at least 15continuous nucleotides in the nucleotide sequence of SEQ ID NO: 1, NO:3, or NO: 5. The above-mentioned antisense oligonucleotide, whichcontains an initiation codon in the above-mentioned at least 15continuous nucleotides, is even more preferred.

The antisense oligonucleotides includes analogs containing lower alkylphosphonate (e.g., methyl-phosphonate or ethyl-phosphonate),phosphothioate, and phosphoamidate.

Herein, the target cells may be mammalian cells, preferably human cells.

The introduction method may be in vitro, in vivo, or ex vivo transfermethod. In one embodiment, the antisense oligonucleotides can beintroduced into the target cells by a conventional transfection method.Alternatively, the introduction can be made by conventional genetransfer technique using a vector carrying the antisenseoligonucleotide, such as adenovirus vectors, retrovirus vectors, orcationic liposomes.

Any patents, patent applications, and publications cited herein areincorporated by reference.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1 a-1 b show expression of a gene termed B9362 in HCCs. FIG. 1 ashows relative expression ratio (cancer/non-cancer) of B9362 in primary20 HCCs examined by cDNA microarray. FIG. 1 b presents photographsshowing expression of B9362 analyzed by semi-quantitative RT-PCR usingadditional 11 HCC cases. Expression of GAPDH served as an internalcontrol.

FIGS. 2 a-2 d show the results of identification of DDEFL1. FIG. 2 a isa photograph showing the results of Northern blot analysis of DDEFL1 invarious human tissues. FIG. 2 b shows the structure of DDEFL1. FIG. 2 cshows similarity between the expected DDEFL1 protein and members ofArfGAP family. FIG. 2 d shows identity between the amino acid sequenceof the ArfGAP motif in DDEFL1 and that in DDEF2. The arrows indicate aCXXCX₁₆CXXC motif, representing a zinc finger structure essential to GAPactivity.

FIGS. 3 a-3 b show subcellular localization of DDEFL1. FIG. 3 a is aphotograph showing the results of Western blot analysis, indicating thatcMyc-tagged DDEFL1 protein was expressed in COS7 cells transfected withpcDNA-DDEFL1-myc plasmid. FIG. 3 b presents photographs showingimmunocytochemistry of the cells, suggesting that cMyc-tagged DDEFL1protein localized in the cytoplasm.

FIGS. 4 a-4 d show growth-promoting effect of DDEFL1. FIG. 4 a presentsphotographs showing the results of colony formation assays, indicatingthat DDEFL1 promotes cell growth in NIH3T3, SNU423, and Alexander cells.FIG. 4 b presents photographs showing stable expression of exogeneousDDEFL1 by NIH3T3-DDEFL1 cells. FIG. 4 c is a graph showing growth ofNIH3T3-DDEFL1 cells stably expressing exogeneous DDEFL1 in culture mediacontaining 10% FBS. FIG. 4 d is a graph showing growth of NIH3T3-DDEFL1cells in culture media containing 0.1% FBS (P<0.01).

FIGS. 5 a-5 b show growth suppression by antisense S-oligonucleotidesdesignated to suppress DDEFL1 in SNU475 cells. FIG. 5 a showsdesignation of antisense S-oligonucleotides and photographs showingreduced expression of DDEFL1 by the transfection of AS1 or AS5 antisenseS-oligonucleotides. FIG. 5 b presents photographs showing that AS1 andAS5 suppressed growth of SNU475 cells.

FIGS. 6 a-6 b show expression of VANGL1 in HCCs. FIG. 6 a shows relativeexpression ratios (cancer/non-cancer) of VANGL1 in primary 20 HCCsexamined by cDNA microarray. FIG. 6 b presents photographs showingexpression of D3244 analyzed by semi-quantitative RT-PCR usingadditional 10 HCC cases. T, tumor tissue; N, normal tissue. Expressionof GAPDH served as an internal control.

FIGS. 7 a and 7B show the results of identification of VANGL1. FIG. 7 ais a photograph showing the results of multiple-tissue Northern blotanalysis of VANGL1 in various human tissues. FIG. 7 b shows predictedprotein structure of VANGL1.

FIGS. 8 a and 8 b show subcellular localization of VANGL1. FIG. 8 apresents photographs of SNU475 cells transfected withpcDNA3.1-myc/His-VANGL1 stained with mouse anti-myc monoclonal antibodyand visualized by Rhodamine conjugated secondary anti-mouse IgGantibody. Nuclei were counter-stained with DAPI. FIG. 8 b presentsphotographs of mock cells similarly stained and visualized.

FIGS. 9 a-9 d show growth suppressive effect of antisenseS-oligonucleotide designated to suppress VANGL1. FIG. 9 a presentsphotographs showing expression of VANGL1 in SNU475 cells treated witheither control or antisense oligonucleotide for 12 hours. FIG. 9 b is aphotograph showing that S-oligonucleotide suppressed growth of SNU423cells. FIG. 9 c is a graph showing the results of analysis of cellviability by MTT assay. FIG. 9 d shows the results of fluorescenceactivated cell sorting (FACS) analysis of cells treated with sense orantisense oligonucleotide.

FIGS. 10 a and 10 b show LGN gene expression of HCCs compared with theircorresponding non-cancerous liver tissues. FIG. 10 a shows relativeexpression ratios (cancer/non-cancer) of LGN in primary 20 HCCs studiedby cDNA microarray. FIG. 10 b presents photographs showing expression ofLGN analyzed by semi-quantitative RT-PCR using additional ten HCCs.Expression of GAPDH served as an internal control. T, tumor tissue; N,normal tissue.

FIG. 11 shows genomic structure of LGN.

FIGS. 12 a-12 c show subcellular localization of LGN. FIG. 12 a is aphotograph of COS7 cells transfected with pcDNA3.1-myc/His-LGN, in whichnuclei was counter-stained with DAPI. FIG. 12 b is a photograph of COS7cells transfected with pcDNA3.1-myc/His-LGN, which were stained withmouse anti c-myc antibody and visualized by Rhodamine conjugatedsecondary anti-mouse IgG antibody. FIG. 12 c is a merge of a and b.

FIGS. 13 a and 13 b show growth-promoting effect of LGN. FIG. 13 apresents photographs showing the results of colony formation assays,indicating that LGN promotes cell growth in NIH3T3, SNU423, Alexander,and SNU475 cells. FIG. 13 b is a graph showing growth of NIH3T3-LGNcells stably expressing exogeneous LGN was higher than that of mock(NIH3T3-LacZ) cells in culture media containing 10% FBS.

FIGS. 14 a and 14 b show growth suppression by antisenseS-oligonucleotide designated to suppress LGN expression in humanhepatoma SNU423 cells. FIG. 14 a presents photographs showing reducedexpression of LGN by the transfection of antisense S-oligonucleotide,antisense 3. FIG. 4 b is a photograph showing that antisense 3suppressed growth of SNU423 cells.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be illustrated with reference to thefollowing examples, but is not construed as being limited thereto.

EXAMPLE 1 1-1. Identification of DDEFL1 Commonly Up-Regulated in HumanHepatocellular Carcinomas

By means of a genome-wide cDNA microarray containing 23040 genes,expression profiles of 20 hepatocellular carcinomas (HCC) were comparedwith their corresponding non-cancerous liver tissues. All HCC tissuesand corresponding non-cancerous tissues were obtained with informedconsent from surgical specimens of patients who underwent hepatectomy. Agene with an in-house accession number of B9362 corresponding to an EST,Hs.44579 of a UniGene cluster, was found to be over-expressed in a rangebetween 1.57 and 5.83 (FIG. 1 a). Its up-regulated expression (Cy3:Cy5intensity ratio, >2.0) was observed in 11 of the 12 HCCs that passedthrough the cutoff filter (both Cy3 and Cy5 signals greater than25,000). Since an open reading frame of this gene encoded a proteinapproximately 60% identical to that of development and differentiationenhancing factor 2 (DDEF2) this gene was termed development anddifferentiation enhancing factor-like 1 (DDEFL1). To clarify the resultsof the cDNA microarray, expression of this transcript was examined in anadditional 11 HCCs by semi-quantitative RT-PCR. Expression of GAPDHserved as an internal control. RT-PCR was performed as follows. TotalRNA was extracted with a Qiagen RNeasy kit (Qiagen) or Trizol reagent(Life Technologies, Inc.) according to the manufacturers' protocols.Ten-microgram aliquots of total RNA were reversely transcribed forsingle-stranded cDNAs using poly dT₁₂₋₁₈ primer (Amersham PharmaciaBiotech) with Superscript II reverse transcriptase (Life Technologies).Single-stranded cDNA preparation was diluted for subsequent PCRamplification by standard RT-PCR experiments carried out in 20-μlvolumes of PCR buffer (TAKARA). Amplification proceeded for 4 min at 94°C. for denaturing, followed by 20 (for GAPDH) or 33 (for DDEFL1) cyclesof 94° C. for 30 s, 56° C. for 30 s, and 72° C. for 45 s, in the GeneAmpPCR system 9700 (Perkin-Elmer, Foster City, Calif.). Primer sequenceswere; for GAPDH: forward, 5′-ACAACAGCCTCAAGATCATCAG (SEQ ID NO: 7) andreverse, 5′-GCTCCACCACTGACACGTTG (SEQ ID NO: 8); for DDEFL1: forward,5′-AGCTGAGACATTTGTTCTCTTG (SEQ ID NO: 9)

and reverse: 5′-TATAAACCAGCTGAGTCCAGAG (SEQ ID NO: 10). The resultsconfirmed increased expression of DDEFL1 in nine of these tumors (FIG. 1b).

1-2. Isolation and Structure of a Novel Gene DDEFL1

Expression of DDEFL1 was analyzed by multiple-tissue northern-blotanalysis using a PCR product of DDEFL1 as a probe. Human multiple-tissueblots (Clontech, Palo Alto., CA) were hybridized with a P-labeled DDEFL1cDNA. Pre-hybridization, hybridization and washing were performedaccording to the supplier's recommendations. The blots wereautoradiographed with intensifying screens at −80° C. for 72 h. Theresults revealed a 4-kb transcript that was expressed in lung, liver,small intestine, placenta and peripheral blood leukocyte (FIG. 2 a).

Since B9362 was smaller than that detected on the Northern blot, 5′RACEexperiments was carried out to determine the entire coding sequence ofthe gene. 5′ RACE experiments were carried out using a Marathon cDNAamplification kit (Clontech, Palo Alto, Calif.) according to themanufacturer's instructions. For the amplification of the 5′ part ofDDEFL1 cDNAs, gene-specific reverse primers (5′-CTCACTTGGCACGTCAGCAGGG(SEQ ID NO: 11)) and the AP-1 primer supplied in the kit were used. ThecDNA template was synthesized from human liver mRNA. The PCR productswere cloned using a TA cloning kit (Invitrogen) and their sequences weredetermined with an ABI PRISM 3700 DNA sequencer (Applied Biosystems).

The complete cDNA consisted of 4050 nucleotides, with an open readingframe of 2712 nucleotides encoding a 903-amino-acid protein (GenBankaccession number AB051853). The first ATG was flanked by a sequence(CCCGCCATGC (SEQ ID NO: 12)) that agreed with the consensus sequence forinitiation of translation in eukaryotes, with an in-frame stop codonupstream. The BLAST program to search for homologies in the NCBI (theNational Center for Biotechnology Information) database, identified agenomic sequence with GenBank accession number AL357134, which had beenassigned to chromosomal band 1p36.12. Comparison of the cDNA and genomicsequences disclosed that DDEFL1 consisted of 25 exons (FIG. 2 b).

A search for protein motifs with the Simple Modular ArchitectureResearch Tool (SMART) revealed that the predicted protein contained twocoiled-coil regions (codons 141-172 and 241-278), a PH (Pleckstrinhomology) motif (codons 303-396), a motif of ArfGAP (GTPase-activatingprotein for Arf) (codons 426-551) and two ankyrin repeats (codons585-617 and 621-653). This structure was similar to centaurin beta 1 andcentaurin beta 2 (FIG. 2 c). In particular, DDEFL1 shared features ofcentaurin-family proteins such as a PH domain, a target ofphosphatidylinositol 3,4,5-trisphosphate, and a motif of ArfGAP. Theamino acid sequence of the ArfGAP motif of DDEFL1 was 67.8% identical tothat of DDEF2 (FIG. 2 d). Notably, the CXXCX₁₆CXXC motif, representing azinc finger structure essential to GAP activity, was completelypreserved.

1-3. Subcellular Localization of DDEFL1

The coding sequence of DDEFL1 was cloned into the pcDNA3.1-myc/H isvector (Invitrogen). The resulting plasmid expressing myc-tagged DDEFL1protein (pDNA-myc/His-DDEFL1) was transiently transfected into COS7cells (American Type Culture Collection (ATCC)). The expected myc-taggedprotein was detected by immunoblotting (Western blotting) as follows.Cells transfected with pcDNA3.1-myc/His-DDEFL1 were washed twice withPBS and harvested in lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mMTris-HCl pH 7.4, 1 mM DTT, and 1× complete Protease Inhibitor Cocktail(Boehringer)). After the cells were homogenized and centrifuged at10,000×g for 30 min, the supernatant was standardized for proteinconcentration by the Bradford assay (Bio-Rad). Proteins were separatedby 10% SDS-PAGE and immunoblotted with mouse anti-myc antibody.HRP-conjugated goat anti-mouse IgG (Amersham) served as the secondaryantibody for the ECL Detection System (Amersham). As a result, theDDEFL1 protein was detected on western blots with an anti-myc antibody(FIG. 3 a).

Furthermore, immunocytochemical staining was performed as follows.First, the cells were fixed with PBS containing 4% paraformaldehyde for15 min. then rendered permeable with PBS containing 0.1% Triton X-100for 2.5 min at RT. Subsequently the cells were covered with 2% BSA inPBS for 24 h at 4° C. to block non-specific hybridization. Mouseanti-myc monoclonal antibody (Sigma) at 1:1000 dilution or mouseanti-FLAG antibody (Sigma) at 1:2000 dilution was used for the firstantibody, and the reaction was visualized after incubation withRhodamine-conjugated anti-mouse second antibody (Leinco and ICN). Nucleiwere counter-stained with 4′,6′-diamidine-2′-phenylindoledihydrochloride (DAPI). Fluorescent images were obtained under anECLIPSE E800 microscope. The microscopic analysis indicated that theprotein was present mainly in the cytoplasm (FIG. 3 b). DDEFL1 was alsolocalized in the cytoplasm of human embryonal kidney (HEK293) cells(ATCC).

1-4. Effect of DDEFL1 on Cell Growth

The coding sequence of DDEFL1 was cloned into the pcDNA 3.1 vector(Invitrogen) NIH3T3 cells (ATCC) plated onto 10-cm dishes (2×10⁵cells/dish) were transfected with the resulting plasmid expressingDDEFL1 (pcDNA-DDEFL1) and the control plasmid (pcDNA-LacZ) and culturedin Dulbecco's modified Eagle's medium supplemented with 10% fetal bovineserum and 1% antibiotic/antimycotic solution (Sigma), and further withan appropriate concentration of geneticin for two weeks. The cells werethen fixed with 100% methanol and stained by Giemsa solution. Cellstransfected with pcDNA-DDEFL1 produced markedly more colonies thancontrol cells. An increase in colony formation similarly occurred withtransfected human hepatoma SNU423 (Korea cell-line bank) and Alexander(ATCC) cells, in which endogenous expression of DDEFL1 is very low (FIG.4 a).

To investigate this growth-promoting effect further, NIH3T3 cells thatstably expressed exogeneous DDEFL1 were established. pDNA-myc/His-DDEFL1was transfected into NIH3T3 cells using FuGENE6 reagent (Boehringer)according to the supplier's recommendations. Twenty-four hours aftertransfection, geneticin was added to the cultures and single colonieswere selected two weeks after transfection. Expression of DDEFL1 wasdetermined by semi-quantitative RT-PCR (FIG. 4 b). The growth rate ofNIH3T3-DDEFL1 cells was statistically higher than that of mock(NIH3T3-LacZ) cells in culture media containing 10% FBS (P<0.05) (FIG. 4c). In media containing only 0.1% FBS, NIH3T3-DDEFL1 cells survived for6 days, while control NIH3T3 cells died within 6 days under the sameconditions. In this case, growth of NIH3T3-DDEFL1 cells wasstatistically higher than that of mock cells in culture media containing0.1% FBS (P<0.01) (FIG. 4 d).

1-5. Suppression of DDEFL1 Expression in Human Hepatoma SNU475 Cells byAntisense S-Oligonucleotides

The following six pairs of control (sense) and antisenseS-oligonucleotides corresponding to the DDEFL1 gene were synthesized.

Antisense: (SEQ ID NO: 13) DDEFL1-AS1 5′-TGCTCCGGCATGGCGG-3′; (SEQ IDNO: 14) DDEFL1-AS2 5′-GCTGAACTGCTCCGGC-3′; (SEQ ID NO: 15) DDEFL1-AS35′-TCCAAGATCTCCTCCC-3′; (SEQ ID NO: 16) DDEFL1-AS45′-TCTCCTTCCAAGATCT-3′; (SEQ ID NO: 17) DDEFL1-AS55′-GCGCTGAGCCGGCCTC-3′; and (SEQ ID NO: 18) DDEFL1-AS65′-CCTCACCTCCTCCCGC-3′. Control: (SEQ ID NO: 19) DDEFL1-S15′-CCGCCATGCCGGAGCA-3′; (SEQ ID NO: 20) DDEFL1-S25′-GCCGGAGCAGTTCAGC-3′; (SEQ ID NO: 21) DDEFL1-S35′-GGGAGGAGATCTTGGA-3′; (SEQ ID NO: 22) DDEFL1-S45′-AGATCTTGGAAGGAGA-3′; (SEQ ID NO: 23) DDEFL1-S55′-GAGGCCGGCTCAGCGC-3′; and (SEQ ID NO: 24) DDEFL1-S65′-GCGGGAGGAGGTGAGG-3′.

Using LIPOFECTIN Reagent (GIBCO BRL), the synthetic S-oligonucleotideswere transfected into SNU475 cells (Korea cell-line bank), which hadshown the highest level of DDEFL1 expression among the six hepatoma celllines we examined (data not shown). Twelve and twenty-four hours aftertransfection, antisense S-oligonucleotides AS1 and AS5 significantlysuppressed expression of DDEFL1 compared to the respective controlS-oligonucleotides S1 and S5 (FIG. 5 a). Six days after transfection,surviving cells transfected with antisense S-oligonucleotide AS1 or AS5were markedly fewer than cells transfected with the respective controlS-oligonucleotide S1 or S5 (FIG. 5 b). Consistent results were obtainedin three independent experiments.

EXAMPLE 2 2-1. Identification of VANGL1 Commonly Up-Regulated in HumanHepatocellular Carcinomas

The genome-wide cDNA microarray analysis carried out in Example 1 alsorevealed that a gene with an in-house accession number of D3244corresponding to an EST (Hs. 122730) of a UniGene cluster, was found tobe significantly up-regulated in ten of twelve clinical HCCs comparedwith the corresponding non-cancerous liver tissues. The relativeexpression ratio compared to corresponding non-cancerous tissue of these12 tumors ranged from 1.5 to 16.0 (FIG. 6 a). Up-regulated expression(Cy3:Cy5 intensity ratio, >2.0) was observed in 10 of the 12 HCCs thatpassed through the cutoff filter (both Cy3 and Cy5 signals greater than25,000). The elevated expression of D3244 was also confirmed in tenadditional HCC cases by semi-quantitative RT-PCR performed similarly toExample 1-1 using the primer set, forward: 5′-GAGTTGTATTATGAAGAGGCCGA(SEQ ID NO: 25); reverse: 5′-ATGTCTCAGACTGTAAGCGAAGG (SEQ ID NO: 26)(FIG. 6 b).

2-2. Expression of VANGL1 in Human Adult Tissues

Multi-tissue northern blot analysis using D3244 cDNA as a probe wasperformed in the same manner as in Example 1-2 and the results showed a1.9-kb transcript abundantly expressed in testis and ovary in atissue-specific manner (FIG. 7 a). NCBI database search for genomicsequences corresponding to D3244 found two sequences (GenBank accessionnumber: AL450389 and AL592436) assigned to chromosomal band 1p22. UsingGENSCAN, and Gene Recognition and Assembly Internet Link program,candidate-exon sequences were predicted and exon-connection wasperformed. In addition, 5′ RACE was carried out using a reverse primer(5′-TGTCAGCTCTCCGCTTGCGGAAAAAAAG (SEQ ID NO: 27)) to determine thesequence of the 5′ region of the transcript in the same manner as inExample 1-2. As a result, an assembled human cDNA sequence of 1879nucleotides containing an open reading frame of 1572 nucleotides(GenBank accession number: AB057596) was obtained. The predicted aminoacid sequence shared 40% and 63% identity with strabismus (Van Gogh) andVANGL2. Hence, the gene corresponding D3244 was termed as Van Gogh Like1 (VANGL1). Simple Modular Architecture Research Tool suggested that thepredicted protein contained putative four transmembrane domains (codons111-133, 148-170, 182-204, 219-241) (FIG. 7 b)

2-3. Subcellular Localization of VANGL1

The pcDNA3.1-myc/His-VANGL1 plasmid expressing c-myc-tagged VANGL1protein was transiently transfected into SNU475 cells (Korea cell-linebank). Immunocytochemical staining was performed in the same manner asin Example 1-3. The results revealed that the tagged VANGL1 protein waspresent in the cytoplasm (FIGS. 8 a and 8 b).

2-4. Growth Suppression of Hepatoma Cells by AntisenseS-Oligonucleotides Designated to Reduce Expression of VANGL1

To test whether suppression of VANGL1 may result in cell cycle arrestand/or cell death of HCC cells, the following four pairs of antisenseand control (sense) S-oligonucleotides were synthesized and transfectedinto SNU475 cells.

Antisense: (SEQ ID NO: 28) antisense 1 5′-GTATCCATAGCAATGG-3′; (SEQ IDNO: 29) antisense 2 5′-TGGATTGGGTATCCAT-3′; (SEQ ID NO: 30) antisense 35′-TAAGTGGATTGGGTAT-3; and (SEQ ID NO: 31) antisense 45′-ACTCCTACCTGCCTGT-3′. Control: (SEQ ID NO: 32) sense 15′-CCATTGCTATGGATAC-3′; (SEQ ID NO: 33) sense 2 5′-ATGGATACCCAATCCA-3′;(SEQ ID NO: 34) sense 3 5′-ATACCCAATCCACTTA-3′; and (SEQ ID NO: 35)sense 4 5′-ACAGGCAGGTAGGAGT-3′.

Antisense S-oligonucleotide encompassing the initiation codon (antisense3) significantly decreased endogenous expression of VANGL1 in SNU475cells (FIG. 9 a).

Cell viability was evaluated by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assayas follows. Cells were plated at a density of 5×10⁵ cells/100 mm dish.At 24 hours after seeding, the cells were transfected in triplicate withsense or antisense S-oligonucleotide designated to suppress VANGL1. At72 hours after transfection, the medium was replaced with fresh mediumcontaining 500 μg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) and the plates were incubated for fourhours at 37° C. Subsequently, the cells were lysed by the addition of 1ml of 0.01 N HCl/10% SDS and absorbance of lysates was measured with anELISA plate reader at a test wavelength of 570 nm (reference, 630 nm).The cell viability was represented by the absorbance compared to that ofcontrol cells.

Transfection of the antisense S-oligonucleotide, antisense 3,significantly reduced number of surviving cells compared with controlsense S-oligonucleotide, sense 3 (FIGS. 9 b and 9 c). This result wasconfirmed by three independent experiments.

Furthermore, flow cytometry analysis was performed as follows. Cellswere plated at a density of 1×10⁵ cells/100 mm dish and trypsinized atthe given time course, followed by fixation in 70% cold ethanol. AfterRNase treatment, cells were stained with propidium iodide (50 μg/ml) inPBS. Flow cytometry was performed on a Becton Dickinson FACScan andanalyzed by CellQuest and ModFit software (Verity Software House). Thepercentages of nuclei in G0/G1, S and G2/M phases of the cell cycle, andany sub-G1 population were determined from at least 20,000 ungatedcells.

FACS analysis demonstrated that inhibition of VANGL1 significantlyincreased number of cells at sub-G1 phase (FIG. 9 d). These resultssuggest that VANGL1 may play an important role for cell growth and/orsurvival of hepatocellular carcinoma cells.

EXAMPLE 3 3-1. LGN is Commonly Increased in Human HepatocellularCarcinomas

Among commonly up-regulated genes by the microarray analysis performedin Example 1-1, a gene, D3636 corresponding to LGN (GenBank accessionnumber: U54999) was selected because it was significantly up-regulatedin ten of twelve clinical HCCs compared with the correspondingnon-cancerous liver tissues. The relative expression ratio compared tocorresponding non-cancerous tissue of these 12 tumors ranged from 0.7 to16.0. Up-regulated expression of LGN (Cy3:Cy5 intensity ratio, >2.0) wasobserved in 10 of the 12 HCCs that passed through the cutoff filter(both Cy3 and Cy5 signals greater than 25,000) (FIG. 10 a). The elevatedexpression of LGN was also confirmed in additional ten HCC cases bysemi-quantitative RT-PCR performed using a primer set, forward:5′-ATCTGAAGCACTTAGCAATTGC (SEQ ID NO: 36), reverse:5′-CTGTAGCTCAGACCAAGAACC (SEQ ID NO: 37), similarly to Example 1-1 (FIG.10 b).

3-2. Genomic Structure of LGN

LGN cDNA consists of 2,336 nucleotides and encodes a 677 amino acidpeptide. Comparison of the cDNA sequence with genomic sequencesdisclosed that the LGN gene consists of 14 exons (FIG. 11).

3-3. Subcellular Localization of LGN

The pcDNA3.1-myc/His-LGN plasmid expressing c-myc-tagged LGN protein wastransiently transfected into COS7 cells. A 72 kDa-band corresponding tomyc-tagged LGN protein was detected by immunoblot analysis in the samemanner as in Example 1-3 (FIG. 12). Similarly, immunocytochemicalstaining was performed as in Example 1-3 and the results revealed thatthe tagged LGN protein was present in the cytoplasm and nucleus in thecells.

3-4. LGN Gene Transfer can Promote Cell Growth

To analyze the effect of LGN on cell growth, a colony-formation assaywas carried out as in Example 1-4 by transfecting NIH3T3, SNU423,Alexander and SNU475 cells with a plasmid expressing LGN(pcDNA3.1-myc/His-LGN). Compared with a control plasmid(pcDNA3.1-myc/His-LacZ), pcDNA3.1-myc/His-LGN produced markedly a largernumber of colonies in these cells (FIG. 13 a). This result was confirmedby three independent experiments.

To further investigate the effect of LGN on cell growth, NIH3T3 cellsthat stably expressed exogeneous LGN(NIH3T3-LGN cells) were established.NIH3T3-LGN cells showed higher growth rate than control NIH3T3-LacZcells (FIG. 13 b).

3-5. Antisense S-Oligonucleotides of LGN Suppressed Growth of HumanHepatoma SNU475 Cells

The following five pairs of control (sense) and antisenseS-oligonucleotides corresponding to LGN were synthesized and thentransfected into SNU423 cells.

Antisense: (SEQ ID NO: 38) antisense 1 5′-CCATCGAGTCATATTA-3′; (SEQ IDNO: 39) antisense 2 5′-TTCCTCCATCGAGTCA-3′; (SEQ ID NO: 40) antisense 35′-AAATTTTCCTCCATCG-3′; (SEQ ID NO: 41) antisense 45′-AGTCTTACCTGTAACG-3′; and (SEQ ID NO: 42) antisense 55′-GCTTCCATTCTACAAA-3′. Sense: (SEQ ID NO: 43) sense 15′-TAATATGACTCGATGG-3′; (SEQ ID NO: 44) sense 2 5′-TGACTCGATGGAGGAA-3′;(SEQ ID NO: 45) sense 3 5′-CGATGGAGGAAAATTT-3′; (SEQ ID NO: 46) sense 45′-CGTTACAGGTAAGACT-3′; and (SEQ ID NO: 47) sense 55′-TTTGTAGAATGGAAGC-3′.

The antisense S-oligonucleotide encompassing the initiation codon(antisense 3) significantly suppressed expression of LGN compared tocontrol S-oligonucleotide (sense 3) 12 hours after transfection (FIG. 14a). Six days after transfection, the number of surviving cellstransfected with antisense 3 were markedly fewer than that with controlsense 3 (FIG. 14 b). Consistent results were obtained in threeindependent experiments.

INDUSTRIAL APPLICABILITY

The present invention provides cDNA nucleotide sequences and polypeptideamino acid sequence of DDEFL1, VANGL1 or LGN, which have been found tobe commonly up-regulated in hepatocellular carcinomas. Thus, thesepolypeptides can be used as markers to determine the presence or absenceof liver cancers. The information of these nucleotide sequences enablesone to design probes and primers to detector amplify the DDEFL1, VANGL1or LGN genes. It also enables synthesis of antisense nucleotide sequencethat inhibits expression of the DDEFL1, VANGL1 or LGN polypeptides. Theamino acid sequence information enables one to prepare antibodies thatbind to the DDEFL1, VANGL1 or LGN polypeptides. The probes and primersas well as the antibodies are useful as a reagent for detectinghepatocellular carcinomas. Furthermore, the present inventorsdemonstrated that suppressing the expression of DDEFL1, VANGL1 or LGN byantisense oligonucleotides markedly decreases growth of HCC cells. Thus,the antisense oligonucleotides can be used to inhibit growth of HCCcells. The present invention also contributes to further clarify themechanisms of hepatocellular carcinogenesis and to discover moleculartargets for development of effective drugs to treat liver cancers.

1. A method for detecting hepatocellular carcinoma, the methodcomprising the steps of: (a) preparing a tissue or cell sample samplefrom a subject; (b) measuring the expression level of the polypeptide ofSEQ ID NO: 4; (c) comparing the expression level with that measured in anon-cancerous sample; and (d) wherein an increase in expressionindicates the presence of hepatocellular carcinoma in the subject.